Download Limited infection without evidence of replication by porcine

Journal of General Virology (2004), 85, 15–19
Short
Communication
DOI 10.1099/vir.0.19495-0
Limited infection without evidence of replication by
porcine endogenous retrovirus in guinea pigs
Takele Argaw, Winston Colon-Moran and Carolyn A. Wilson
Laboratory of Immunology and Virology, Division of Cellular and Gene Therapies, Center for
Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike,
HFM-725, Bethesda, MD 20892, USA
Correspondence
Carolyn Wilson
[email protected]
Received 8 July 2003
Accepted 30 September 2003
Porcine endogenous retrovirus (PERV) may potentially be transmitted through porcine
xenotransplantation products administered to humans. This study examined the feasibility of
using guinea pigs as a model to characterize the in vivo infectivity of PERV. To enhance the
susceptibility of guinea pigs to retroviral infection or genomic integration, moderate physiological
or immunological changes were induced prior to exposing the animals to PERV. Quantitative
PERV-specific PCR performed on all tested samples resulted in either undetectable or very low
copy numbers of proviruses, even in animals possessing PERV-specific antibody responses. The
low copy number of viral DNA detected suggests that PERV infected a limited number of cells.
However, PERV DNA levels did not increase over time, suggesting no virus replication occurred.
These results in the guinea pig are similar to previous observations of non-human primate cells
that allow PERV infection but do not support PERV replication in vitro.
The supply of human organs is insufficient to meet the
demand for transplantation and this has led some members
of the scientific and medical communities to consider use of
organs from animals, particularly from the pig (Lai et al.,
2002; Tucker et al., 2002). Although data from clinical trials
in pig-to-human xenotransplantation have not provided
evidence for transmission of porcine endogenous retrovirus
(PERV) (Loss et al., 2001; Paradis et al., 1999; Patience et al.,
1998; Switzer et al., 2001), the possibility of cross-species
transmission of PERV as well as other pig-derived infectious
agents is still of concern in xenotransplantation procedures.
PERV is a gammaretrovirus present in the pig genome in
multiple copies (Todaro et al., 1974). Several studies have
demonstrated that porcine primary cells and continuous cell
lines can release PERV virions that replicate in cells from pig,
cat, mink and human (Martin et al., 2000; Patience et al.,
1997; Takeuchi et al., 1998; Wilson et al., 1998). However, no
studies, retrospective or prospective, have found evidence
for transmission of PERV from porcine xenotransplantation
products to human recipients (Heneine et al., 1998; Paradis
et al., 1999; Patience et al., 1998; Schumacher et al., 2000;
Tacke et al., 2001). Additionally, studies of PERV performed
in small animals and non-human primates have been unsuccessful in finding a model that supports PERV replication
(Specke et al., 2001; Switzer et al., 2001). The notable exceptions have been two studies in immunodeficient mice
implanted with porcine islets, where evidence of limited
PERV transmission to murine tissues was observed (Deng
et al., 2000; van der Laan et al., 2000). In contrast, unpublished data presented by Dr David Onions at a meeting of the
FDA’s Subcommittee on Xenotransplantation: 13 January
0001-9495 G 2004 SGM
2000 (Xenotransplantation Committee, 2000) provided
evidence from an animal model where up to 70 000 copies
of PERV DNA were detected in the spleen of guinea pigs
several weeks after exposure to PERV in an immunization
protocol. This preliminary finding suggested that guinea
pigs might support active virus replication, in contrast to the
negative results reported by Specke et al. (2001). However,
there were some key experimental differences in how the
animals were treated in these two studies. In particular, the
unpublished data were based on an experiment meant to
immunize the animals, while the study by Specke and
coworkers did not use an immunizing strategy. Therefore,
we sought to examine whether and under what circumstances guinea pigs may provide a feasible model to assess
the in vivo replication properties of PERV.
In the studies described here, outbred strain Hartley guinea
pigs (HARLAN), 2–3 weeks old, were used. The Institutional
Animal Care and Use Committee (CBER/FDA) reviewed
and approved all experiments.
The virus isolate used in all experiments was PERV-NIH,
derived originally from NIH mini pigs as previously described (Wilson et al., 1998). Serial passage through HEK 293
cells resulted in a virus producer line capable of generating
viral titres of >105 ml21. The MoMLV-based retroviral
vector genome G1BgSvN (McLachlin et al., 1993), encoding
the bacterial lacZ gene, was introduced to generate the virus
used in our experiments: PERV-NIH-169 b-gal. These cells
produce a mixture of PERV-NIH virions and pseudovirions
composed of PERV-NIH core and envelope surrounding
the MoMLV-based G1BgSvN genome. The presence of the
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15
T. Argaw, W. Colon-Moran and C. A. Wilson
G1BgSvN genome allows use of histochemical staining
for lacZ activity to determine the infectious titre of viral
stocks used in this study as previously described (Wilson &
Eiden, 1991).
In a preliminary study two groups of animals (n=16) were
exposed to 26106 PERV-NIH producer cells or viruscontaining supernatant. These animals were killed at different time points post-inoculation (up to a total of 4 weeks
post-initial inoculation). Gross examination upon necropsy
did not reveal any pathological changes. Genomic DNA was
isolated from tissues and subjected to PCR analysis combined with Southern blotting (Wilson et al., 1998). Similar
to the results of Specke et al. (2001), only rare positive results
were obtained, without consistent positive results at different time points (data not shown). These results indicated
that intact guinea pigs were not readily infected with PERV;
we therefore sought to determine other experimental
conditions that may enhance the susceptibility of guinea
pigs to PERV infection.
Chemical stimulation or mild liver damage induces hepatocyte proliferation, increasing susceptibility to retroviral
vector infection (Forbes et al., 1998; Kitten et al., 1997). To
assess whether a similar regimen would increase susceptibility to PERV, we exposed guinea pigs to allyl alcohol (AA)
(0?05 ml kg21 intraperitoneally, i.p.), an agent known to
induce mild liver necrosis (Werlich et al., 1999; Yin et al.,
1999) prior to i.p. inoculation of PERV. Animals were
inoculated i.p. with two doses of virus (46105 blueforming units, b.f.u.) at 24 and 48 h after AA administration. At each end time point (see Fig. 1) four animals were
killed. As controls, two animals inoculated with PERV,
without AA treatment, and one animal inoculated with
complete DMEM only, were killed at each end time point.
To measure the effectiveness of AA in inducing hepatocyte
proliferation, we administered 50 mg kg21 of BrdU (Sigma)
to guinea pigs (n=16) 24 and 48 h post AA injection and
2 h prior to killing. Samples of liver tissues were randomly
excised during postmortem examination and preserved
for histochemistry and histopathological analysis. Liver sections (6–10 mm) were obtained from American HistoLabs
(Gaithersburg, MD, USA) and subjected to immunohistochemical staining using a BrdU in situ detection kit (BD,
BioSciences PharMinegen). The staining for BrdU-positive
cells, as a measure of proliferative response, revealed a mean
of 7?9±2?2 % positive hepatocytes in liver samples obtained
from AA-treated animals killed 3 days post viral exposure
(n=4) compared to 1?6±0?4 % positive hepatocytes in
liver samples obtained from untreated animals (n=2). Additionally, a preliminary study performed on three animals to
assess the effect of AA treatment on liver function resulted
in a 100–115 % and 55–60 % increase in blood level of liver
enzymes (aspartate transaminase and alanine transaminase)
at 24 h and 48 h post AA administration, respectively,
relative to untreated controls, indicating liver damage.
PERV pol-specific real-time quantitative PCR (qPCR) was
performed, as previously described (Argaw et al., 2002), on
16
Fig. 1. Experimental design for guinea pig infectivity studies.
(A) Induction of hepatocyte proliferation with AA followed by
two exposures to virus; (B) enhancing the immunity to PERV by
injecting disrupted virus mixed with CFA. Treatments are shown
as indicated: D, day; Wk, week; AA, allyl alcohol; V, virus; No,
no treatment; CFA, complete Freund’s adjuvant. All inoculations
of virus only were i.p.
genomic DNA (gDNA) isolated from tissue samples
obtained from animals killed at the indicated time points
post-treatment. Within each tissue type, the analysis yielded
variable results, ranging from 0 to 433 copies of viral DNA
per 100 ng gDNA tested. Generally, the highest detectable
copy numbers of the virus and the greatest number of
PERV-positive tissues per animal were observed at early
time points, 3–7 days post viral exposure, decreasing with
later time points (Table 1). Treatment of guinea pigs with
AA did not induce an increase in infectivity in animals
with evidence of liver damage, although in other studies
mild and transient liver damage has resulted in proliferation
of hepatocytes that eased retrovirus cellular transduction
and augmented transgene expression (Kitten et al., 1997;
Rettinger et al., 1994).
We next performed a study based on the unpublished results
of D. Onions (Xenotransplantation Committee, 2000) to see
whether an initial immunizing dose of PERV might influence the outcome of infection when animals were later
inoculated with live PERV. While typically immunization
would be expected to prevent or reduce infectivity, it has
also been postulated that virus-specific antibodies may
enhance infectivity (Morens, 1994; Olsen, 1993). The previous PERV study in guinea pigs (Xenotransplantation
Committee, 2000) detected 3000–70 000 copies of viral
DNA per 106 cells in spleen tissue isolated from guinea pigs
immunized with complete Freund’s adjuvant (CFA)-treated
PERV followed by a booster dose with live PERV. To
determine whether an anti-PERV antibody response may
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Journal of General Virology 85
Limited PERV infection of guinea pigs
Table 1. Quantitative PCR analysis and biodistribution of PERV DNA in AA-treated and untreated animals
Mean copies of viral DNA per 100 ng gDNA in tissuesD
Time post first
virus injection
Treatment*
Animal no.
3 days
AA+PERV
ap31
ap32
ap33
ap34
p31
p32
ap71
ap72
ap73
ap74
p71
p72
ap151
ap152
ap153
ap154
p151
p152
ap301
ap302
ap303
ap304
p301
p302
PERV only
7 days
AA+PERV
PERV only
15 days
AA+PERV
PERV only
30 days
AA+PERV
PERV only
L
S
K
Ln
In
Pb
6
2
3
162
12
32
6
72
8
11
26
433
23
25
2
21
21
27
26
15
3
0
3
0
32
4
11
30
8
23
4
17
8
9
88
17
23
11
2
7
2
4
1
5
0
0
0
0
57
25
22
60
22
3
3
4
7
4
0
31
18
19
3
1
10
28
2
4
0
0
0
0
17
23
8
130
17
23
4
3
7
5
0
189
6
31
4
5
5
0
5
0
0
0
0
0
4
16
25
24
11
1
2
1
15
7
4
217
15
2
1
5
16
15
3
2
0
0
0
0
0
0
0
2
1
1
0
0
1
0
0
3
4
2
0
0
2
0
6
3
0
0
0
0
*Animals were exposed to two doses of PERV (46105 infectious virions) at 24 and 48 h after AA (0?05 ml kg21) treatment. One negative control
animal was allocated for each end time point and i.p. inoculation of DMEM in lieu of PERV after AA treatement. The indicated tissues were also
harvested from the negative control animals and tested for PERV. No PERV viral DNA was detected in the tissues from negative control animals.
gDNA isolated from the negative control animals served as assay background, and was also spiked with a known concentration of PERV plasmid
DNA and tested for any possible inhibitory effect.
DL, liver; S, spleen; K, kidney; Ln, lymph node; In, intestine; Pb, PBMC.
enhance virus replication, we first immunized guinea pigs to
PERV by inoculating subcutaneously with an equal volume
mixture of CFA and PERV virions (56105 b.f.u.). Four
weeks later, animals were inoculated with the same dose level
of live PERV in the absence of adjuvant. Animals were bled
and killed at the time points indicated in Table 2 and Fig. 1,
to collect serum and different tissue samples for immunoassay and DNA extraction. While low levels of viral DNA
were detected in some samples at 8 and 12 weeks after the
initial immunization (4 and 8 weeks after live PERV inoculation), no DNA was detected in any samples examined from
animals killed 16 weeks post-inoculation (Table 2).
Guinea pig plasma samples were analysed for the presence
of PERV-specific antibodies by Western blot assay against
whole virion lysate, as previously described (Tacke et al.,
2001). Anti-SSAV p30 goat serum (lot 81S000315, NCI
repository, Quality Biotech) served as a positive control to
run the immunoassay. Analysis of anti-p27 PERV antibody
demonstrated that the immunization strategy was effective:
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specific anti-PERV antibodies were detected in animals
inoculated with PERV, but not in control uninoculated
animals (data not shown). Immunological modulation has
also been reported to enhance the infectivity of some viruses
via antibody-dependent enhancement (ADE) of infection
(reviewed by Morens, 1994; Olsen, 1993) whereby the entry
of virus–antibody complexes into cells results in significantly increased virus infectivity. This phenomenon has
been experimentally correlated with enhanced infectivity of
several viruses, including dengue virus (Morens & Halstead,
1990), Ross river virus (Lidbury & Mahalingam, 2000), HIV
(Kozlowski et al., 1995), and feline infectious peritonitis
virus (Olsen, 1993). In our experiment, animals were
immunized with PERV mixed with CFA, to determine
whether ADE of PERV infection occurs. However, we
observed that presence of anti-PERV antibodies did not
enhance PERV infection of guinea pigs, as demonstrated by
the lack of differences in viral DNA copy number per tissue
detected in animals treated with CFA or untreated prior to
inoculation with live PERV (Table 2).
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T. Argaw, W. Colon-Moran and C. A. Wilson
Table 2. Tissue distribution of PERV pol DNA in tissues of animals exposed to virus (56105 infectious virions) after immunization with PERV and complete Freund’s adjuvant (CFA) subcutaneously
Mean copies of viral DNA per 100 ng gDNA from tissuesD
Time post first
virus injection
Treatment*
Animal no.
8 weeks
CFA+PERV
12 weeks
PERV
CFA+PERV
16 weeks
PERV
CFA+PERV
Fa81
Fa82
Fa83
P8
Fa121
Fa122
Fa123
Fa124
P12
Fa161
Fa162
Fa163
Fa164
P160
PERV
L
S
K
Ln
I
T/O
Pb
AG
14
8
12
6
3
2
7
11
4
0
0
0
0
1
5
0
16
4
2
3
3
5
1
0
0
1
0
0
9
0
7
9
2
3
12
8
2
0
0
0
0
0
9
0
15
3
7
10
2
3
0
0
0
2
0
2
8
11
0
2
5
2
10
10
2
0
0
0
0
1
0
6
11
8
1
22
7
2
0
3
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
–
–
–
–
5
2
8
15
4
0
0
0
0
0
*Treatments were adjuvant (CFA) plus PERV followed by inoculation with live PERV only, or inoculation with PERV only without prior
adjuvant treatment. One negative control animal was allocated for each end time point and was i.p. inoculated with DMEM. The indicated tissues
were also harvested from the negative control animals and tested for PERV. No PERV viral DNA was detected in the tissues from negative control
animals. gDNA isolated from the negative control animals served as assay background, and was also spiked with a known concentration of PERV
plasmid DNA and tested for any possible inhibitory effect.
DL, liver; S, spleen; K, kidney; Ln, lymph node; I, intestine; T/O, testis/ovary; Pb, PBMC; AG, adrenal gland.
Viral inoculation of naı̈ve animals may lead to persistent,
transient, or no evidence of infection (Jilbert et al., 1998;
Zinkernagel, 1996). We found that exposure of guinea pigs
to PERV-NIH produces only a transient low-level viral
infection, as measured by detection of viral DNA most consistently at early time points, and with decreased frequency
and copy number at later time points after viral inoculation
(Tables 1, and 2). In addition, gross examination of organs
during necropsy and limited histopathological examinations did not reveal any virus-induced gross or microscopic
lesions in any animals examined.
The detection of PERV viral DNA in some tissues indicates
that PERV may be able to infect guinea pig cells. Either
tightly controlled suppression of virus replication or a
potent host clearance mechanism against PERV may explain
the reduced levels of viral DNA detected at later time points.
The latter interpretation is supported by the durable
humoral immunity observed in animals (data not shown)
during the time-course of the experiment (16 weeks).
In a recent study of non-human primate cells exposed to
PERV, we found that PERV infection was restricted, resulting in low copy numbers of viral DNA and lack of virus
replication (Ritzhaupt et al., 2002). The results reported here
from the guinea pig studies, where only a low copy number
of viral DNA was detected, implies that a similar mechanism
restricting virus replication may operate in guinea pigs.
Although it is out of the scope of this study, the adaptation
18
of viral stocks by serial passage in guinea pig tissues might
increase the replication capacity of PERV.
Apart from two reports (Deng et al., 2000; van der Laan et al.,
2000) showing limited transmission of PERV to immunodeficient mice, no other conventional laboratory small
animal had been previously well investigated for susceptibility to PERV infection. After a preliminary study on intact
animals, we have investigated whether immunological or
physiological manipulations can increase susceptibility to
PERV replication in guinea pigs. Our study clearly demonstrates that guinea pigs are refractory to PERV replication.
ACKNOWLEDGEMENTS
We thank Oliver Zill and Elizabeth Skelton for providing expert technical assistance. We are also indebted to the animal care unit of the
Division of Veterinary Service of CBER/FDA for their valuable laboratory animal services and excellent animal care. We thank Drs Eda Bloom
and Steven Bauer for manuscript review and valuable comments.
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